Review: Sperm cryopreservation in wild small ruminants: morphometric, endocrine and molecular basis of cryoresistance.

Reproductive technologies can help to protect wild ruminant species from becoming extinct. In addition, the decline in some wild game species has also raised interest in reproductive technologies to increase the number of animals that can be produced. Most biobanking efforts have focused on developing effective protocols for preserving sperm, oocytes, and embryos. Cryopreservation of sperm remains the least invasive method and the cheapest procedure for germplasm storage. Over the last few years, several reproductive biotechnologies have been developed beyond the conventional freezing of spermatozoa. These include ultra-rapid freezing techniques. Nevertheless, fertility results after artificial insemination using frozen-thawed spermatozoa are not always acceptable in wild small ruminants. Moreover, these technological efforts have met variable success related to the sample's origin (epididymal retrieved postmortem or ejaculated) and the season of sperm sample collection and storage. Epididymal sperm shows higher cryoresistance than ejaculated sperm. Changes in sperm proteome between epididymal and ejaculated sperm seem to contribute to this different cryotolerance. The role of endocrine status has been studied in some wild species to better understand the underlying mechanism of the annual variation in ruminant sperm cryoresistance. Seasonal changes in testosterone and prolactin are involved in sperm cryoresistance; sperm recovery and cryopreservation are recommended around the end of the rutting season, when good quality sperm samples can still be obtained, testosterone levels have already decreased, and prolactin concentrations remain low. The mechanisms of hormone action on sperm freezability are not well known. Still, it has been suggested that testosterone affects cell proliferation in the testis, during spermatogenesis, and membrane properties of sperm cells during their transit through the reproductive tract, which might influence their cryotolerance. Recent studies have revealed that the expression of aquaporins in the sperm cells of small wild ruminants could also be involved in the androgen-related seasonal variation of sperm cryoresistance. Along with epididymal and ejaculated spermatozoa, the cryopreservation of testicular tissue may provide a suitable source of male gametes, becoming an alternative for establishing germplasm banks when semen cannot be collected for whatever reason.

Use of the flavonoid taxifolin for sperm cryopreservation from the threatened Bermeya goat breed.

Taxifolin is a plant flavonoid effective as an antioxidant. This study aimed to assess the effect of adding taxifolin to the semen extender during the cooling period before freezing on the overall post-thawing sperm variables of Bermeya goats. In the first experiment, a dose-response experiment was performed with four experimental groups: Control, 10, 50, and 100 µg/ml of taxifolin using semen from 8 Bermeya males. In the second experiment, semen from 7 Bermeya bucks was collected and extended at 20 °C using a Tris-citric acid-glucose medium supplemented with different concentrations of taxifolin and glutathione (GSH): control, 5 µM taxifolin, 1 mM GSH, and both antioxidants. In both experiments, two straws per buck were thawed in a water bath (37 °C, 30 s), pooled, and incubated at 38 °C. Motility (CASA) was assessed at 0, 2, and 5 h, and sperm physiology was assessed at 0 and 5 h by flow cytometry (viability, intact acrosome membrane, mitochondria membrane potential, capacitation, intracellular reactive oxygen species -ROS-, mitochondrial superoxide, and chromatin status). In experiment 2, an artificial insemination trial (AI) was included with 29 goats for testing the taxifolin 5-µM treatment on fertility. Data were analyzed with the R statistical environment using linear mixed-effects models. In experiment 1 and compared to the control, T10 increased progressive motility (P < 0.001) but taxifolin decreased total and progressive motility at higher concentrations (P < 0.001), both post-thawing and after the incubation. Viability decreased post-thawing in the three concentrations (P < 0.001). Cytoplasmic ROS decreased at 0 and 5 h at T10 (P = 0.049), and all doses decreased mitochondrial superoxide post-thawing (P = 0.024). In experiment 2, 5 µM taxifolin or 1 mM GSH (alone or combined) increased total and progressive motility vs. the control (P < 0.01), and taxifolin increased kinematic parameters such as VCL, ALH, and DNC (P < 0.05). Viability was not affected by taxifolin in this experiment. Both antioxidants did not significantly affect other sperm physiology parameters. The incubation significantly affected all the parameters (P < 0.004), overall decreasing sperm quality. Fertility after artificial insemination with doses supplemented with 5 µM taxifolin was 76.9% (10/13), not significantly different from the control group (69.2%, 9/13). In conclusion, taxifolin showed a lack of toxicity in the low micromolar range and could benefit goat semen cryopreservation.

Cryopreservation of testicular tissue from the dog (Canis familiaris) and wild boar (Sus scrofa) by slow freezing and vitrification: Differences in cryoresistance according to cell type.

Sperm cryopreservation is the most common procedure used to establish germplasm banks for endangered species - but sometimes sperm cells cannot be obtained. In such cases, freezing testicular tissue may be the only option. The testes contains germ cells at different stages of differentiation, including spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and spermatozoa, among which differences in cryoresistance might be expected. The present work compares the viability and DNA integrity of 'rounded' cells, and of elongated spermatids and spermatozoa, from the dog and wild boar, following the cryopreservation of testicular tissue by slow freezing or vitrification. Cell viability was analyzed by PI/SYBR14 staining, and DNA integrity via the TUNEL technique. For wild boar, no significant differences were seen between the two methods with respect to the percentage of viable cells, nor in the percentage of cells with DNA damage. In the dog, the percentage of viable rounded germ cells (65.0 ± 2.4%) was higher (P < 0.05) after vitrification than after slow freezing (45.1 ± 6.7%). No difference was found between the two methods in terms of the viability of elongated cells. For rounded cells, the percentage of intact DNA was greater (P < 0.05) after vitrification (90.5 ± 2.1%) than after slow freezing (42.6 ± 11.0%), while for elongated spermatids and spermatozoa it was higher (P < 0.05) after slow freezing (66.9 ± 6.1%) than after vitrification (50.7 ± 4.5%). Thus, the response to cryopreservation is cell type-, cryopreservation type-, and species-dependent. Vitrification would appear to be the most appropriate method for preserving dog testicular tissue given the associated high cell viability and low degree of DNA fragmentation, while in wild boar, either method might be used.

Transit along the vas deferens results in a high percentage of filiform spermatozoa with compacted chromatin in the rooster (Gallus domesticus).

The present work aimed to evaluate the chromatin compaction of rooster spermatozoa along the male reproductive tract, and to study the vas deferens lining cells, potentially involved in sperm maturation. Chromomycin A3 (CMA3) was used to determine the chromatin compaction of spermatozoa from testis (T), proximal (including epididymis, V1), intermediate (V2) and distal (V3) vas deferens, and ejaculate (E). Six Birchen Leonesa roosters were used. E was obtained in vivo by dorso-ventral massage. V1, V2 and V3 sperm were obtained post mortem (six pairs of vasa deferentia), by flushing. T was obtained by washing the testes, cut in halves. The fixed cells were stained with CMA3 and propidium iodide for flow cytometry assessment. Results showed higher (P P P.

Optimization of semen cryopreservation in black-footed (Spheniscus demersus) and gentoo (Pygoscelis papua) penguins using dimethylacetamide and dimethylsulfoxide.

Black-footed penguins (Spheniscus demersus) are classified as endangered, and the populations of gentoo penguins (Pygoscelis papua) are rapidly decreasing. The optimization of semen cryopreservation in these species, for preserving their genetic diversity in genome resource banks, is essential for the success of captive breeding programs. This study compares the effectiveness of two permeating cryoprotectants, dimethylacetamide (DMA) and dimethylsulfoxide (DMSO), on frozen-thawed sperm characteristics. Semen samples were collected during each breeding season once a week during two consecutive years. Semen samples were packaged in 0.25 ml straws and frozen by placing them in nitrogen vapors. After thawing, sperm motility characteristics were examined by computer-assisted sperm analysis. Propidium iodide and SYBR-14 were used as fluorochromes for the examination of membrane integrity. DNA integrity was evaluated by TUNEL assay. Gentoo sperm characteristics after freeze-thawing did not show any differences when using DMSO or DMA. In black-footed samples, progressive motility, curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), and straightness (STR) were greater using 8% DMSO (P < 0.05) than 6% DMA. The cryoresistance ratio (CR) using 8% DMSO was greater (P < 0.05) in gentoo than black-footed samples for CR-VCL and CR-VAP, and 6% DMA returned greater CR values (P < 0.05) than in black-footed samples for all characteristics evaluated. No differences were found in DNA fragmentation. In conclusion, the present results highlight the benefits of using 8% DMSO compared to 6% DMA in penguins. Sperm from black-footed showed a higher sensitivity to freezing-thawing process than gentoo sperm.

Birchen and Blue Leonesa sperm cryopreservation: a new technique for evaluating the integrity of cockerel sperm membranes.

1. Birchen and Blue Leonesa are two endangered chicken breeds mainly raised in Curueño Valley in North Spain. The establishment of a germplasm bank to guarantee the preservation of these breeds is needed. However, cockerels from different breeder flocks can show variance in semen cryoresistance.2. The following work focused on the sperm characterisation and cryopreservation of Birchen and Blue Leonesa cockerels from four different breeders. A total of 30 semen pools were analysed. Besides conventional sperm analysis, including motility by computer-aided sperm analysis (CASA) and DNA fragmentation by TUNEL, the present study tested a double staining method (MitoTrackerTM Green FM/propidium iodide). This gave simultaneous assessment of plasma and acrosomal and mitochondrial membranes, which were previously validated by SYBR-14/PI, CASA, aniline blue and TUNEL.3. No significant differences were found among fresh semen variables between breeds and breeders. For post-thawed variables, significant differences (P < 0.05) were found between breeders in sperm viability (58.0 ± 1.90 breeder D vs. 35.2 ± 7.41 breeder A, 37.2 ± 4.09 breeder B and 22.3 ± 5.92 breeder C) and DNA fragmentation (62.4 ± 9.91 breeder C vs. 31.8 ± 7.08 breeder B and 24.5 ± 5.49 breeder D). The lowest DNA fragmentation values for semen from breeder D birds were coincident with higher integrity of the mitochondrial membrane.4. The results revealed higher sperm cryoresistance in the cockerels from one of the breeders, possibly due to differences in management system (e.g. diet, housing, control of stress elements and pathogens, reproduction practices or maintenance of genetic diversity). These differences may determine the sperm freezability, and thus the effectiveness of developing a germplasm bank.

Vitrification of Iberian wolf (Canis lupus signatus) sperm: A possible alternative to conventional cryopreservation.

Sperm vitrification is a simple, inexpensive method that allows the cryopreservation of sperm in the field and for endangered species is a useful alternative to conventional freezing. The study, therefore, is focused on the suitability of vitrification for cryopreserving Iberian wolf sperm and utilizing plasma testosterone concentration as a marker for procedure efficacy. Sperm and blood samples were collected from 17 wolves. There were 14 samples suitable for cryopreservation (12 ejaculated and two epididymal). Immediately after collection, these samples were proportioned into two aliquots for conventional freezing using a Tris-citric acid-glucose based extender (TCG) or vitrification utilizing an animal protein free extender (HTF®). Vitrification occurred by directly plunging a sperm suspension into liquid nitrogen. Sperm were assessed for motility, membrane integrity, acrosomal status and DNA integrity before and after cryopreservation. With both techniques, there were similar post-thaw/warming results (P > 0.05) with respect to progressive motility, kinetic variables VCL, VSL, VAP and BCF, DNA fragmentation, sperm membrane functionality and morphological abnormalities. Total motile sperm, progression ratios LIN, STR, and WOB, the ALH, sperm viability and sperm with intact membrane and acrosome were greater (P < 0.05) in the conventional frozen-thawed sperm than vitrified-warmed sperm. Plasma testosterone concentrations varied from 0.0 ng/mL to 7.7 ng/mL. For epididymal sperm, sperm motility and viability following thawing were greater in vitrified-warmed samples than conventionally-frozen samples; however, small sample numbers precluded statistical analysis. When considered together, these results indicate vitrification may be a possible alternative for wolf sperm cryopreservation.

Role of changes in plasma prolactin concentrations on ram and buck sperm cryoresistance.

Seasonal endocrine changes may modify sperm cryoresistance in certain small ruminant species. The present work examines the effect of prolactin (PRL) on ram and buck sperm cryoresistance. A dopamine agonist (bromocriptine [BCR] 60 mg i.m. twice per week from May 15 to June 15, that is, approaching the summer solstice) or antagonist (sulpiride [SLP] 100 mg s.c. daily from December 15 to January 15, that is, around the winter solstice) was administered under solstice-appropriate photoperiod conditions to modify PRL secretion. Control animals received the vehicle only. Compared to the corresponding controls, BCR reduced PRL secretion to basal levels in both the rams and bucks. In rams, the cryoresistance ratios for sperm curvilinear velocity (P < 0.05) and lateral head displacement (P < 0.01) were higher for the BCR-treated animals. In bucks, neither the characteristics of fresh nor frozen-thawed sperm were affected by BCR treatment. After the administration of SLP, PRL levels increased and remained high for more than 5 h in the rams though they immediately began to fall in the bucks. By 24 h, PRL had returned to basal concentrations in both species. In rams treated with SLP, the cryoresistance ratios for sperm progressive motility, straight line velocity, sperm mean path velocity, cross beat frequency, and the progression ratios linearity, straightness and oscillation, were all lower compared to the controls (P < 0.05), while the amplitude of lateral head displacement was higher (P < 0.01). In bucks, sperm cryoresistance was not affected by SLP administration. Together, these results suggest that high levels of PRL negatively affect the cryoresistance of ram sperm, while buck sperm seems unaffected.

Effect of supplementation of valine to chicken extender on sperm cryoresistance and post-thaw fertilization capacity.

Recent reports showed a positive correlation between frozen-thawed rooster sperm DNA integrity and the concentrations of valine in seminal plasma. The present study evaluated the effect of supplementing valine to semen extender for freezing sperm of 2 endangered local Spanish chicken breeds with different sperm cryoresistance: Red Villafranquina (VF) showing low sperm DNA integrity after cryopreservation and Quail Castellana that shows higher DNA integrity. One pool of semen per breed was obtained twice a week for 10 wk (n = 40, 20 per breed). Each pool was divided into 2 fractions. One of these fractions was frozen in presence of valine as additive in the extender (concentration 10 mmol), whereas the other was used as control. The evaluation of the samples before and after freezing-thawing included motility (CASA-Mot system), viability (propidium iodide and SYBR-14), DNA integrity (terminal deoxynucleotidyl transferase dUTP nick end labeling), and fertility rate (percentage of eggs with blastoderm development after artificial insemination). Supplementation of valine increased several motility variables of fresh semen. In VF breed, valine increased percentage of progressive motile sperm (P = 0.025), curvilinear velocity (P = 0.033), straight-line velocity (P = 0.040), and average path velocity (P = 0.033), whereas progressive motile sperm (P = 0.019), curvilinear velocity (P = 0.006), straight-line velocity (P = 0.003) and average path velocity (P = 0.004) were improved in the Quail Castellana breed. Valine addition increased the DNA integrity of cryopreserved semen (decreased post-thaw DNA fragmentation) in both breeds, with a significant effect (P = 0.002) in VF (36.3% VF-control vs 31%VF-valine). As expected, Quail Castellana cryopreserved sperm control showed higher fertility rate (34.4% ± 12.1) than VF cryopreserved sperm control (16.1% + 6.2). Supplementing valine to the freezing extender doubled (P = 0.026) the fertility rate of VF (32.6% ± 12.2) compared with the control (16.1% + 6.2). In conclusion, supplementation of valine to chicken freezing extenders shows a positive effect on DNA fragmentation and fertilizing ability of frozen-thawed sperm, with a better response in a breed considered as the lowest freezer in our conservatory.

Influence of testosterone administration at the end of the breeding season on sperm cryoresistance in rams (Ovis aries) and bucks (Capra hircus).

This study examines the influence of administering testosterone at the end of the mating season, on the responses (morphometric and functional) of ram and buck sperm to freezing-thawing. Five rams were administered 25 mg testosterone propionate (TP) subcutaneously in 2 mL of olive oil twice per week (Monday and Thursday) from October 1 to 31; 5 bucks received exactly the same treatment but from November 1 to 30. Control groups were administered 2 mL of olive oil without TP twice per week over the same period. In the rams, no significant differences were seen in plasma testosterone between the treated and control groups during treatment (0.8 ± 0.2 ng/mL vs 1.5 ± 0.5 ng/mL; P > 0.05). Significant differences were seen in this respect, however, in the bucks (4.3 ± 0.8 ng/mL and 6.9 ± 0.9 ng/mL; P < 0.05). In the rams, TP treatment increased (P < 0.05) the straight-line velocity (VSL), linearity (LIN), straightness (STR) and wobble (WOB) values in fresh sperm samples. Similarly, in the frozen-thawed samples, TP treatment increased the VSL, average path velocity (VAP), LIN and WOB values (P < 0.05) compared with controls. In the bucks, treatment with TP had no effect on any measured variable in fresh sperm; frozen-thawed sperm, however, returned greater VSL, LIN, STR, and WOB values (P < 0.05) than did controls. In the rams, treatment with TP led to a reduction in all fresh sperm head morphometric variables (P < 0.05). Freezing-thawing further reduced (P < 0.05) all morphometric variables in both the control and treated groups. In the bucks, treatment with TP increased (P < 0.05) the length, area, and perimeter of fresh sperm cells, unlike that seen in ram sperm. Compared with fresh sperm, freezing-thawing led to reduced (P < 0.05) morphometric variables in both the control and treated bucks, except for the sperm head width, which in the controls remained unchanged. In conclusion, TP treatment at the end of the mating season affected fresh sperm quality, in both Spanish Merino rams and Murciano-Granadina bucks, in a species-specific manner, but improved the sperm kinetic variables after freezing-thawing in both species, apparently improving sperm cryoresistance. Treatment with TP affects the dimensions of the sperm head in a species-specific manner.

In vitro supplementation of testosterone or prolactin affects spermatozoa freezability in small ruminants.

In small ruminants, testosterone and prolactin plasma concentrations show circannual fluctuations as an adaptation mechanism to their seasonal breeding behavior. Sperm resistance to the freezing-thawing process shows seasonal fluctuation throughout the year, with lower sperm freezability at the beginning of the breeding season when prolactin and testosterone levels reach their maximum concentration. Nevertheless, whether these hormones directly affect post-thaw sperm quality parameters is still unclear. The objective was to study the effect of testosterone or prolactin added in vitro on sperm freezability in domestic ram (Ovis aries) and buck (Capra hircus). Sperm samples were incubated for 1 h with a range of testosterone (0, 2, 4, or 6 ng/mL; Exp. 1) or prolactin (0, 20, 100, 200, or 400 ng/mL; Exp. 2) concentrations. Samples were cryopreserved by slow freezing in straws at 0 h and after 1 h incubation. Sperm viability, acrosome integrity, motility, and kinetic parameters were assessed at 0 and 1 h in fresh and frozen-thawed samples. Results showed no hormone effect in fresh sperm, whereas these hormones affected post-thaw sperm parameters. In Exp. 1, in vitro incubation with testosterone decreased the post-thaw acrosome integrity of ram sperm (from 68.1 ± 6.3% to 49.6 ± 3.9%; P < 0.05). In Exp. 2, in vitro incubation with prolactin decreased the post-thaw acrosome integrity of ram (from 78.2 ± 3.4% to 66.3 ± 3.5%; P < 0.05) and buck sperm (from 81.7 ± 2.5% to 67.6 ± 3.5%; P < 0.05). Moreover, prolactin increased the post-thaw amplitude of lateral head displacement in ram sperm (from 3.3 ± 0.1 µm to 3.8 ± 0.2 µm; P < 0.05). In conclusion, either testosterone or prolactin added in vitro decreased the post-thaw acrosome integrity of ram and buck sperm. This suggests a destabilization process that could be decreasing sperm freezability when physiological levels of these hormones are high in vivo.

Effectiveness of ultra-rapid cryopreservation of sperm from endangered species, examined by morphometric means.

This study compares the effectiveness of the ultra-rapid and conventional freezing of sperm from captive bovids, giraffids, cervids, ursids, a cercopithecid, a delphinid and a phascolarctid. The relationship between sperm head dimensions and cryosurvival was also examined. Compared to conventional freezing, the ultra-rapid freezing of epididymal sperm from the dama gazelle, giraffe and brown bear returned higher cryoresistance ratios (CR, the ratio, in percentage, between the value of the variable after thawing/value before thawing) for sperm viability and motility. In the remaining species, the conventional freezing of epididymal sperm returned better CR values. The conventional freezing method also returned better CR values for ejaculated samples from all species. The head dimensions of both fresh epididymal and ejaculated sperm differed widely among species: for epididymal sperm, dolphin sperm heads were the smallest (7.189 ±â€¯0.049 µm2) and dama gazelle sperm heads the largest (43.746 ±â€¯0.291 µm2), while for ejaculated sperm, giant panda sperm heads were the smallest (15.926 ±â€¯0.150 µm2) and mouflon sperm heads the largest (38.258 ±â€¯0.104 µm2). However, no significant correlations were detected between the CR for motility, viability, membrane functional integrity or acrosome integrity and the sperm head area, either for epididymal or ejaculated sperm. In conclusion, ultra-rapid freezing is especially recommended for the cryopreservation of dama gazelle, giraffe and brown bear epididymal sperm. Sperm head dimensions appear not to be useful predictors of how well sperm might survive freezing.

Semen cryopreservation in black-footed (Spheniscus demersus) and gentoo (Pygoscelis papua) penguins: Effects of thawing temperature on semen characteristics.

In this study, there was an examination of the effect on the characteristics of cryopreserved black-footed (Spheniscus demersus) and gentoo (Pygoscelis papua) penguin semen, of thawing at 37 and 5 °C. For two consecutive years, semen was collected and frozen during the April-June period from six gentoo penguins, and during the October-November period from 13 black-footed penguins. After thawing, sperm motility variables were examined by computer-assisted sperm analysis. Propidium iodide and SYBR-14 were used as fluorochromes for the examination of membrane integrity. For the gentoo penguins, no differences were detected in the values of frozen-thawed semen characteristics after thawing at 37 or 5 °C. For the black-footed penguins, however, thawing at 5 °C resulted in greater values (P < 0.05) for straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), straightness (STR), and wobble (WOB) as compared with thawing at 37 °C. After thawing at 37 ºC, there were greater values with gentoo penguin sperm for percentage motile sperm, progressive motility, curvilinear velocity (VCL), VSL VAP, LIN, STR, WOB and beat-cross frequency (BCF; P < 0.05) than that for black-footed penguin sperm. After thawing at 5 ºC, there were no differences in values for any variables between the two species. In conclusion, thawing temperature affects semen characteristics in a species-specific manner. The present data strongly suggest that cryopreservation procedures should be adapted for use with each penguin species. Cryopreserved black-footed penguin semen should be thawed after cryopreservation at 5 ºC, while that of gentoo penguins can be thawed at either 5 or 37 ºC.

Access to pasture in an outdoor housing system affects welfare indicators and improves rooster sperm quality in two native Mediterranean breeds.

The aim of the present work was to examine the influence of access to pasture in an outdoor housing system on rooster sperm quality and response to cryopreservation and to examine the possible correlation between values for sperm quality variables and welfare indicators. Two groups of Black-barred Andaluza and Red-barred Vasca roosters were housed in an outdoor system, with one group given daily access to a grazing area containing plant species that typically grow on uncultivated Mediterranean land. Semen was collected once per week from each group, and the following sperm quality variables were assessed: sperm volume, appearance, concentration, motility, membrane integrity, acrosome integrity, and morphological abnormalities. In addition, two welfare indicators were examined: the heterophil/lymphocyte (H/L) ratio, and the duration of tonic immobility (TI). Ejaculates from the birds with access to pasture had higher percentages of sperm showing progressive motility (P = 0.019), and returned a higher motility index (P = 0.035). Unexpectedly, the H/L ratio was also higher in these birds. Virtually no differences were seen between the treatment groups with respect to sperm quality after freezing-thawing, although the semen of the Red-barred Vasca birds with access to pasture did show a higher percentage of progressive motility (P = 0.023) than the birds of the same breed with no such access. Significant correlations were detected between the H/L ratio and sperm motility (r = 0.420, P = 0.038), the sperm motility index (r = 0.526, P = 0.002), and progressive motility (r = 0.467, P = 0.003). No differences were seen between the treatment groups with respect to the duration of TI. In conclusion, access to pasture improved fresh sperm motility.

Slow and ultra-rapid freezing protocols for cryopreserving mouflon (Ovis musimon) and fallow deer (Dama dama) epididymal sperm.

This study examines the effectiveness of two methods for cryopreserving post-mortem epididymal sperm - conventional slow freezing employing a short equilibration time with glycerol, and ultra-rapid freezing - from the wild ruminant species Ovis musimon (mouflon) and Dama dama (fallow deer). A Tris-citric acid-glucose (TCG) + 12% egg yolk-based medium was used for the conventional slow freezing of the fallow deer sperm, whereas a Tes-Tris-glucose (TEST) + 6% egg yolk-based medium was used for the mouflon sperm. Glycerol was added to a final concentration of 5% to both media. The same diluents were used for ultra-rapid freezing but replacing the glycerol with 100 mM of sucrose. Sperm variables (motility, viability, acrosome integrity, membrane integrity, and morphological abnormalities) were analyzed before and after cryopreservation. Although values were generally better after the thawing of the conventionally cryopreserved sperm, total sperm motility (38.40 ±â€¯4.44% in mouflon and 31.25 ±â€¯3.37% in fallow deer) and total live sperm (47.19 ±â€¯5.18% in mouflon and 43.13 ±â€¯2.43% in fallow deer) were acceptable for the ultra-rapidly cooled sperm. Independent of the cryopreservation method, membrane integrity, acrosome integrity and the percentages of dead sperm and sperms with a damaged acrosome were better for the cryopreserved mouflon sperm than the fallow deer sperm (P < 0.05). Despite exerting a more harmful effect on sperm variables than conventional freezing, ultra-rapid freezing may be a useful alternative for the cryopreservation of these species' epididymal sperm in the field, as this simple technique does not require sophisticated equipment and expertise.

Successful chilling of red-legged partridge (Alectoris rufa) sperm for use in artificial insemination.

The fertilizing capacity of pure, fresh avian semen may disappear in just half an hour, hindering its successful use in artificial insemination (AI) projects. Longer storage requires the use of infra-physiological temperatures and of semen diluents that help preserve the spermatozoa but that do not interfere with their fertilizing capacity. This study examines the effect on sperm quality of storing red-legged partridge sperm for 3 h at 5°C with 2 different semen extenders: 1) a medium referred to as L&R-84, composed of sodium glutamate, glucose, magnesium acetate, potassium acetate, and polyvinylpyrrolidone, and 2) Lake 7.1 medium, composed of sodium glutamate, glucose, magnesium acetate, potassium citrate, and N,N-Bis(2-hydroxyethyl)taurine (BES). Extending with L&R-84 returned better curvilinear velocity (P < 0.01), straight-line velocity (P < 0.01), average path velocity (P < 0.01), linearity (P < 0.05), straightness (P < 0.05), and wobble (P < 0.05) values, while extending with the Lake 7.1 medium was associated with higher percentages (P < 0.001) of motile sperm. The fertility rate was higher (P < 0.05) when birds were inseminated with L&R-84-extended sperm than with Lake 7.1-extended sperm. The mean number of penetrations of perivitelline layer samples (taken from above the germinal disc) was also higher for the L&R-84-extended sperm (P < 0.05). These results show L&R-84 can be recommended as an extender for red-legged partridge semen to be stored for at least 3 h at 5°C.

Seminal plasma removal by density-gradient centrifugation is superior for goat sperm preservation compared with classical sperm washing.

Seminal plasma removal is routine in goat sperm cryopreservation protocols. The classical washing procedure designed to accomplish this usually leaves the pellet resulting from use of this procedure contaminated with dead sperm, debris, and cells other than sperm. This contamination negatively affects viability of sperm after cryopreservation. The present research was conducted to compare the effect on chilled and frozen-thawed goat sperm of the classical washing method to that of a selective washing method involving density gradient centrifugation (DGC). In the first experiment, sperm variables were measured in freshly collected sperm, and again after its washing with both methods and chilling at 5°C for 0, 3, 24, 48, 72 or 96h. The DGC-washed sperm had greater (P<0.01) straight line velocity (VSL), average path velocity (VAP) and progression ratio values at all chilling times. The amplitude of lateral head displacement (ALH) was, however, less (P<0.001) in the DGC-washed sperm at all chilling times. There was a negative correlation (P<0.05) between ALH and VSL. In the second experiment involving the freezing-thawing of sperm washed by using either method, aliquots were post-wash diluted with a Tris-citric acid/glucose/egg yolk/glycerol-based medium and frozen in liquid nitrogen for 5days. After thawing, neither the VCL, VSL nor VAP of the DGC-washed samples were affected, whereas the traditionally washed samples had less motility. In conclusion, the use of DGC was associated with enhanced sperm motility variables after chilling and freezing-thawing. This procedure would, therefore, be a useful means of removing seminal plasma from goat semen and obtaining greater quality sperm for insemination purposes.

Conventional slow freezing cryopreserves mouflon spermatozoa better than vitrification.

This work examines the effectiveness of a TCG (Tris, citric acid, glucose, 6% egg yolk and 5% glycerol) and a TEST (TES, Tris, glucose, 6% egg yolk and 5% glycerol) sperm extender in the freezing of mouflon spermatozoa at slow cooling rates, using different pre-freezing equilibration times (2-3 hr). It also examines the tolerance of mouflon spermatozoa to different concentrations of cryoprotectants (5, 10, 20% glycerol; 5%, 10%, 20% dimethyl sulfoxide; 6% polyvinylpyrrolidone) and/or sucrose (100, 300, 500 mm). The highest quality (p < .01) thawed spermatozoa were obtained when using the TEST extender and an equilibration time of 3 hr. Sperm motility and membrane integrity were strongly reduced when using rapid freezing rates (60-85°C min-1 ), independent of the concentration of cryoprotectants. The lowest sucrose concentration (100 mm) provided the highest (p < .05) percentage of motile spermatozoa and live spermatozoa with an intact acrosome. Vitrified-warmed sperm variables were at their best when the spermatozoa was diluted in TCG-6% egg yolk + 100 mm sucrose and warmed at 60°C. Slow warming at 37°C strongly reduced (p < .05) sperm motility and viability. However, sperm vitrification returned lower fertility, sperm motility and sperm viability values than conventional sperm freezing.

Effect of shortening the prefreezing equilibration time with glycerol on the quality of chamois (Rupicapra pyrenaica), ibex (Capra pyrenaica), mouflon (Ovis musimon) and aoudad (Ammotragus lervia) ejaculates.

The present study reports the effect of shortening the prefreezing equilibration time with glycerol on the quality of frozen-thawed ejaculated sperm from four Mediterranean mountain ungulates: Cantabrian chamois (Rupicapra pyrenaica), Iberian ibex (Capra pyrenaica), mouflon (Ovis musimon) and aoudad (Ammotragus lervia). Ejaculated sperm from these species were divided into two aliquots. One was diluted with either a Tris-citric acid-glucose based medium (TCG-glycerol; for chamois and ibex sperm) or a Tris-TES-glucose-based medium (TTG-glycerol; for mouflon and aoudad sperm), and maintained at 5°C for 3h prior to freezing. The other aliquot was diluted with either TCG (chamois and ibex sperm) or TTG (mouflon and aoudad sperm) and maintained at 5°C for 1h before adding glycerol (final concentration 5%). After a 15min equilibration period in the presence of glycerol, the samples were frozen. For the ibex, there was enhanced (P<0.05) sperm viability and acrosome integrity after the 3h as compared with the 15min equilibration time. For the chamois, subjective sperm motility and cell membrane functional integrity were less (P<0.05) following 15min of equilibration. In the mouflon, progressive sperm motility and acrosome integrity was less (P<0.05) when the equilibration time was reduced to 15min. For the aoudad, the majority of sperm variables measured were more desirable after the 3h equilibration time. The freezing-thawing processes reduced the sperm head size in all the species studied; however, the equilibration time further affected the frozen-thawed sperm head variables in a species-dependent fashion. While the equilibration time for chamois sperm might be shortened, this appears not to be the case for all ungulates.

Giant panda (Ailuropoda melanoleuca) sperm morphometry and function after repeated freezing and thawing.

This work examines the effects of subsequent cycles of freezing-thawing on giant panda (Ailuropoda melanoleuca) sperm morphometry and function, and assesses whether density-gradient centrifugation (DGC) can increase the number of freezing-thawing cycles this sperm can withstand. A sperm sample was collected by electroejaculation from a mature giant panda and subjected to five freezing-thawing cycles. Although repeated freezing-thawing negatively affected (P < 0.05) sperm motility and membrane integrity, in both nonselected and DCG-selected sperm samples, >60% of the sperm cells in both treatments showed acrosome integrity even after the fifth freezing cycle. In fresh semen, the sperm head length was 4.7 µm, the head width 3.6 µm, area 14.3 µm(2) and perimeter length 14.1 µm. The present results suggest that giant panda sperm trends to be resistant to repeated freezing-thawing, even without DGC selection.